To better understand O-glycosylation and the transport of O-linked glycoproteins to their site of residence, a map of the O-glycosylation events that occur as a transmembrane protein is transported from the endoplasmic reticulum (ER) through the Golgi complex will be generated. This map will then be used to identify the pre-Golgi and Golgi compartments where sugar addition and O-acetylation of sialic acid occur and to determine the role of ras-related GTP binding proteins during transport. The reporter protein to be used is glycophorin (gp3), an exclusively O-glycosylated protein from murine erythroleukemia (MEL) cells. Glycophorin is extensively modified during transport through the Golgi, and changes in its mobility on polyacrylamide gels as it acquires these modifications will be used to assess transport. Partially processed gp3 has been identified in preliminary experiments in which MEL cells were treated with brefeldin A (BFA) to disrupt the Golgi complex and to halt transport at the ER. Following removal of BFA and reassembly of the Golgi, gp3 intermediates appear that do not correspond to known precursor or mature forms of gp3. The specific aims of this project are to: 1) Use HPLC analysis and radiolabeled sugar incorporation to identify the sugar residues on the gp3 intermediates. 2) Identify the pre-Golgi and Golgi compartments where O-glycosyltransferases and O-acetyltransferases are acting. 3) Determine the role of GTP-binding proteins in transport using antibodies, peptides, or mutant proteins corresponding to GTP-binding proteins and assessing their effects on the transport of gp3. 4) Immunolocalize the O-glycosyltransferases and O-acetyltransferase in MEL cells using immunofluorescence and immunogold labeling. Ultimately, this information will be useful for understanding the modified O-glycosylation observed in a number of malignant cells.